Methods of using viral replicase polynucleotides and polypeptides

ABSTRACT

The invention provides novel methods of using viral replicase polypeptides. Included are methods for increasing transformation frequencies, providing a positive growth advantage, and modulating cell division.

[0001] This application is a divisional of co-pending application U.S.Se. No. 09/627,107 filed Jul. 27, 2000, which is a divisional ofco-pending U.S. Pat. No. 6,284,947 filed Feb. 25, 1999; the disclosuresof which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to plant molecularbiology.

BACKGROUND OF THE INVENTION

[0003] Cell division plays a crucial role during all phases of plantdevelopment. The continuation of organogenesis and growth responses to achanging environment requires precise spatial, temporal anddevelopmental regulation of cell division activity in meristems (and incells with the capability to form new meristems such as in lateral rootformation). Such control of cell division is also important in organsthemselves (i.e. separate from meristems per se), for example, in leafexpansion and secondary growth.

[0004] A complex network controls cell proliferation in eukaryotes.Various regulatory pathways communicate environmental constraints, suchas nutrient availability, mitogenic signals such as growth factors orhormones, or developmental cues such as the transition from vegetativeto reproductive. Ultimately, these regulatory pathways control thetiming, frequency (rate), plane and position of cell divisions.

[0005] Plants have unique developmental features that distinguish themfrom other eukaryotes. Plant cells do not migrate, and thus only celldivision, expansion and programmed cell death determine morphogenesis.Organs are formed throughout the entire life span of the plant fromspecialized regions called meristems.

[0006] In addition, many differentiated cells have the potential to bothdedifferentiate and to reenter the cell cycle. The study of plant cellcycle control genes is expected to contribute to the understanding ofthese unique phenomena. O. Shaul et al., Regulation of Cell Division inArabidopsis, Critical Reviews in Plant Sciences 15(2):97-112 (1996).

[0007] Current transformation technology provides an opportunity toengineer plants with desired traits. Major advances in planttransformation have occurred over the last few years. However, in manymajor crop plants, serious genotype limitations still exist.Transformation of some agronomically important crop plants continues tobe both difficult and time consuming.

[0008] For example, it is difficult to obtain a culture response fromsome maize varieties. Typically, a suitable culture response has beenobtained by optimizing medium components and/or explant material andsource. This has led to success in some genotypes. While, transformationof model genotypes is efficient, the process of introgressing transgenesinto production inbreds is laborious, expensive and time consuming. Itwould save considerable time and money if genes could be introduced intoand evaluated directly in commercial hybrids.

[0009] There is evidence to suggest that cells must be dividing fortransformation to occur. It has also been observed that dividing cellsrepresent only a fraction of cells that transiently express a transgene.Furthermore, the presence of damaged DNA in non-plant systems (similarto DNA introduced by particle gun or other physical means) has been welldocumented to rapidly induce cell cycle arrest (W. Siede, Cell cyclearrest in response to DNA damage: lessons from yeast, Mutation Res.337(2):73-84). Methods for increasing the number of dividing cells wouldtherefore provide valuable tools for increasing transformationefficiency.

[0010] Current methods for genetic engineering in maize require aspecific cell type as the recipient of new DNA. These cells are found inrelatively undifferentiated, rapidly growing meristems, in callus, insuspension cultures, or on the scutellar surface of the immature embryo(which gives rise to callus). Irrespective of the delivery methodcurrently used, DNA is introduced into literally thousands of cells, yettransformants are recovered at frequencies of 10⁻⁵ relative totransiently-expressing cells.

[0011] Exacerbating this problem, the trauma that accompanies DNAintroduction directs recipient cells into cell cycle arrest andaccumulating evidence suggests that many of these cells are directedinto apoptosis or programmed cell death. (Reference Bowen et aL., TucsonInternational Mol. Biol. Meetings). Therefore it would be desirable toprovide improved methods capable of increasing transformation efficiencyin a number of cell types.

[0012] In spite of increases in yield and harvested area worldwide, itis predicted that over the next ten years, meeting the demand for cornwill require an additional 20% increase over current production(Dowswell, C. R., Paliwal, R. L., Cantrell, R. P. 1996. Maize in theThird World, Westview Press, Boulder, Colo.).

[0013] The components most often associated with maize productivity aregrain yield or whole-plant harvest for animal feed (in the forms ofsilage, fodder, or stover). Thus the relative growth of the vegetativeor reproductive organs might be preferred, depending on the ultimate useof the crop. Whether the whole plant or the ear are harvested, overallyield will depend strongly on vigor and growth rate. It would thereforebe valuable to develop new methods that contribute to the increase incrop yield.

SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide methods formodulating DNA replication in a transgenic plant.

[0015] It is another object of the present invention to provide a methodfor increasing the number of cells undergoing cell division.

[0016] It is another object of the present invention to provide a methodfor increasing crop yield.

[0017] It is another object of the present invention to provide a methodfor improving transformation frequencies.

[0018] It is another object of the present invention to provide a methodfor improving transformation efficiency in cells from various sources.

[0019] It is another object of the present invention to provide a methodfor providing a positive growth advantage in a plant.

[0020] Therefore, in one aspect, the present invention provides a methodfor increasing transformation frequencies comprising introducing into atarget cell a viral replicase polynucleotide operably linked to apromoter driving expression in the target cell or introducing a viralreplicase polypeptide.

[0021] In another aspect the present invention provides a method forincreasing crop yield comprising introducing into a plant cell anisolated viral replicase polynucleotide operably linked to a promoterdriving expression in the plant cell.

[0022] In another aspect the invention provides a method for providing apositive growth advantage in a target cell comprising introducing intothe target cell an isolated viral replicase polynucleotide operablylinked to a promoter driving expression in the target cell.

[0023] In another aspect the invention provides a method for modulatingcell division of target cells comprising introducing into the targetcell an isolated viral replicase polynucleotide in sense or antisenseorientation operably linked to a promoter driving expression in thetarget cell or introducing an isolated viral replicase polypeptide.

[0024] In another aspect the invention provides a method for transientlymodulating cell division of target cells comprising introducing into thetarget cells an isolated viral replicase polynucleotide in sense orantisense orientation operably linked to a promoter driving expressionin the target cells, an isolated viral replicase polypeptide, or anantibody directed against a viral replicase polypeptide.

[0025] In another aspect the invention provides a method for providing ameans of positive selection comprising (a) introducing into a targetcell an isolated viral replicase polynucleotide operably linked to apromoter driving expression in the target cell or an isolated viralreplicase polypeptide and (b) selecting for cells exhibiting positivegrowth advantage.

DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1: Comparison of transient GUS expression with and without aRep-expression cassette.

[0027] FIG. 2: Micrograph comparison of GFP fluorescence in cellsbombarded with and without a Rep-expression cassette.

[0028] FIG. 3: Comparison of cell cycle profile in callus transformedwith and without a Rep-expression cassette.

DEFINITIONS

[0029] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in its naturally occurring environment or (2) if the material isin its natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in the cellother than the locus native to the material.

[0030] As used herein, “polypeptide” and “protein” are usedinterchangeably and mean proteins, protein fragments, modified proteins,amino acid sequences and synthetic amino acid sequences. The polypeptidecan be glycosylated or not.

[0031] As used here, “polynucleotide” and “nucleic acid” are usedinterchangeably. A polynucleotide can be full-length or a fragment andincludes polynucleotides that have been modified for stability. Unlessotherwise indicated, the term includes reference to a specific sequenceor its complement.

[0032] As used herein, “functional variant” or “functional derivative”or “functional fragment” are used interchangeably. As applied topolypeptides, the functional variant or derivative is a fragment, amodified polypeptide, or a synthetic polypeptide that stimulates DNAreplication in a manner similar to the wild-type gene products, Rep andRepA.

[0033] As used herein, “viral replicase polypeptides” refers topolypeptides capable of stimulating DNA replication. The polypeptidesare intended to include functional variants, fragments, and derivatives.The polypeptides exhibit the function of binding to the family ofretinoblastoma (Rb) proteins, or Rb-associated proteins, or functionalRb homologs. The polypeptides include functional variants or derivativesof viral replicase proteins, and/or functional homologues. Thepolypeptides include proteins encoded by genes in the viral genome thatare commonly referred to as “replication proteins”, “replicationassociated proteins”, or “replication initiation proteins”. Thepolypeptide includes proteins from viruses in which all the “replicationassociated” or “replication” functions are encoded as a single protein,and those in which these functions are carried out by more than oneprotein, irrespective of whether proper or “inappropriate” splicing hasoccurred prior to translation (thus including both the polypeptideencoded by the C1 Open Reading Frame, and the polypeptide encoded by theC1-C2 fusion or properly spliced C1-C2).

[0034] As used herein, “viral replicase polynucleotide” refers topolynucleotides coding for a viral replicase polypeptide, includingfunctional variants, derivatives, fragments, or functional homologs ofcharacterized viral replicase polynucleotides.

[0035] As used herein, “plant” includes but is not limited to plantcells, plant tissue and plant seeds.

[0036] The present invention provides novel methods of using viralreplicase polypeptides and polynucleotides. Included are methods forincreasing transformation frequencies, increasing crop yield, providinga positive growth advantage, modulating cell division, transientlymodulating cell division, and for providing a means of positiveselection.

[0037] Viral replicase polynucleotides, functional variants and/orfunctional homologs from any virus can be used in the methods of theinvention as long as the expressed polypeptides exhibit Rb bindingfunction, and/or stimulates DNA replication.

[0038] Examples of suitable plant viruses include wheat dwarf virus,maize streak virus, tobacco yellow dwarf virus, tomato golden mosaicvirus, abutilon mosaic virus, cassava mosaic virus, beet curly topvirus, bean dwarf mosaic virus, bean golden mosaic virus, chlorisstriate mosaic virus, digitaria streak virus, miscanthus streak virus,maize streak virus, panicum streak virus, potato yellow mosaic virus,squash leaf curl virus, sugarcane streak virus, tomato golden mosaicvirus, tomato leaf curl virus, tomato mottle virus, tobacco yellow dwarfvirus, tomato yellow leaf curl virus, African cassava mosaic virus, andthe bean yellow dwarf virus.

[0039] Other viral proteins that bind Rb-related peptides include thelarge-T antigen from SV40, adenovirus type 5 E1A protein, and humanpapilloma virus type 16 - E7. Replicase from the wheat dwarf virus hasbeen sequenced and functionally characterized and is thereforepreferred. Replicase binds to a well-characterized binding motif on theRb protein (Xie et al., The EMBO Journal Vol. 14, No. 16, pp. 4073-4082,1995; Orozco et al., Journal of Biological Chemistry, Vol. 272, No. 15,pp. 9840-9846, 1997; Timmermans et al., Annual Review Plant Physiology.Plant Mol. Biol, 45:79-112, 1994; Stanley, Genetics and Development3:91-96, 1996; Davies et al., Geminivirus Genomes, Chapter 2, andGutierrez, Plant Biology 1:492-497, 1998). The disclosures of theseitems are incorporated herein by reference.

[0040] Viral replicase polynucleotides useful in the present inventioncan be obtained using (a) standard recombinant methods, (b) synthetictechniques, or combinations thereof.

[0041] Viral replicase polynucleotides and functional variants useful inthe invention can be obtained using primers that selectively hybridizeunder stringent conditions. Primers are generally at least 12 bases inlength and can be as high as 200 bases, but will generally be from 15 to75, preferably from 15 to 50. Functional fragments can be identifiedusing a variety of techniques such as restriction analysis, Southernanalysis, primer extension analysis, and DNA sequence analysis.

[0042] Variants of the nucleic acids can be obtained, for example, byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like. See, forexample, Ausubel, pages 8.0.3-8.5.9. Also, see generally, McPherson(ed.), DIRECTED MUTAGENESIS: A Practical approach, (IRL Press, 1991).Thus, the present invention also encompasses DNA molecules comprisingnucleotide sequences that have substantial sequence similarity with theinventive sequences. Conservatively modified variants are preferred.

[0043] Nucleic acids produced by sequence shuffling of viral replicasepolynucleotides can also be used. Sequence shuffling is described in PCTpublication No. 96/19256. See also, Zhang, J.- H., et al. Proc. Natl.Acad. Sci. USA 94:4504-4509 (1997).

[0044] Also useful are 5′ and/or 3′ UTR regions for modulation oftranslation of heterologous coding sequences. Positive sequence motifsinclude translational initiation consensus sequences (Kozak, NucleicAcids Res.15:8125 (1987)) and the 7-methylguanosine cap structure(Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elementsinclude stable intramolecular 5′ UTR stem-loop structures (Muesing etal., Cell 48:691 (1987)) and AUG sequences or short reading frames 5′ ofthe appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. andCell. Biol. 8:284 (1988)).

[0045] Further, the polypeptide-encoding segments of the polynucleotidescan be modified to alter codon usage. Codon usage in the coding regionsof the polynucleotides of the present invention can be analyzedstatistically using commercially available software packages such as“Codon Preference” available from the University of Wisconsin GeneticsComputer Group (see Devereaux et al., Nucleic Acids Res. 12:387-395(1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).

[0046] For example, the polynucleotides can be optimized for enhanced orsuppressed expression in plants. See, for example, EPA0359472;WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Inthis manner, the genes can be synthesized utilizing species-preferredcodons. See, for example, Murray et al. (1989) Nucleic Acids Res.17:477-498, the disclosure of which is incorporated herein by reference.

[0047] The nucleic acids may conveniently comprise a multi-cloning sitecomprising one or more endonuclease restriction sites inserted into thenucleic acid to aid in isolation of the polynucleotide. Also,translatable sequences may be inserted to aid in the isolation of thetranslated polynucleotide of the present invention. For example, ahexa-histidine marker sequence provides a convenient means to purify theproteins of the present invention.

[0048] The polynucleotides can be attached to a vector, adapter,promoter, transit peptide or linker for cloning and/or expression of apolynucleotide of the present invention. Additional sequences may beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide, or to improve the introduction of the polynucleotideinto a cell. Use of cloning vectors, expression vectors, adapters, andlinkers is well known and extensively described in the art. For adescription of such nucleic acids see, for example, Stratagene CloningSystems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, AmershamLife Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

[0049] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol.152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

[0050] The genomic library can be screened using a probe based upon thesequence of a nucleic acid used in the present invention. Those of skillin the art will appreciate that various degrees of stringency ofhybridization can be employed in the assay; and either the hybridizationor the wash medium can be stringent. The degree of stringency can becontrolled by temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide.

[0051] Typically, stringent hybridization conditions will be those inwhich the salt concentration is less than about 1.5 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

[0052] Preferably the hybridization is conducted under low stringencyconditions which include hybridization with a buffer solution of 30%formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and awash in 1× to 2× SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50°C. More preferably the hybridization is conducted under moderatestringency conditions which include hybridization in 40% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55° C. Mostpreferably the hybridization is conducted under high stringencyconditions which include hybridization in 50% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.1× SSC at 60° C.

[0053] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et aL., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

[0054] The nucleic acids can be amplified from nucleic acid samplesusing amplification techniques. For instance, polymerase chain reaction(PCR) technology can be used to amplify the sequences of polynucleotidesof the present invention and related genes directly from genomic DNA orlibraries. PCR and other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes.

[0055] Examples of techniques useful for in vitro amplification methodsare found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methodsand Applications, Innis et al., Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used toimprove yield of long PCR products.

[0056] PCR-based screening methods have also been described. Wilfingeret al. describe a PCR-based method in which the longest cDNA isidentified in the first step so that incomplete clones can be eliminatedfrom study. BioTechniques, 22(3): 481-486 (1997).

[0057] The nucleic acids can also be prepared by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862 (1981); the solidphase phosphoramidite triester method described by Beaucage andCaruthers, Tetra. Letts. 22(20): 1859-1862 (1981), e.g., using anautomated synthesizer, e.g., as described in Needham-VanDevanter et al.,Nucleic Acids Res., 12:6159-6168 (1984); and, the solid support methodof U.S. Pat. No. 4,458,066.

[0058] Expression cassettes comprising the isolated viral replicasenucleic acids are also provided. An expression cassette will typicallycomprise a polynucleotide operably linked to transcriptional initiationregulatory sequences that will direct the transcription of thepolynucleotide in the intended host cell, such as tissues of atransformed plant.

[0059] The construction of expression cassettes that can be employed inconjunction with the present invention is well known to those of skillin the art in light of the present disclosure. See, e.g., Sambrook etal.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, NewYork; (1989); Gelvin et al.; Plant Molecular Biology Manual; (1990);Plant Biotechnology: Commercial Prospects and Problems, eds. Prakash etal.; Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslotet al.; Molecular Biology and Genetic Engineering of Yeasts; CRC Press,Inc., USA; (1992); each incorporated herein in its entirety byreference.

[0060] For example, plant expression cassettes may include (1) a viralreplicase nucleic acid under the transcriptional control of 5′ and 3′regulatory sequences and (2) a dominant selectable marker. Such plantexpression cassettes may also contain, if desired, a promoter regulatoryregion (e.g., one conferring inducible, constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0061] Constitutive, tissue-preferred or inducible promoters can beemployed. Examples of constitutive promoters include the cauliflowermosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenasepromoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter,the rubisco promoter, the GRP1-8 promoter and other transcriptioninitiation regions from various plant genes known to those of skill.

[0062] Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight. Also useful are promoters which are chemically inducible.

[0063] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruit, seeds, or flowers. An exemplary promoteris the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051). Examples of seed-preferred promoters include, but are notlimited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A.,Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, J.; Isolationand sequencing of a 28 kD glutelin-2 gene from maize: Common elements inthe 5′ flanking regions among zein and glutelin genes; Plant Sci.47:95-102 (1986) and Reina, M., Ponte, I., Guillen, P., Boronat, A. andPalau, J., Sequence analysis of a genomic clone encoding a Zc2 proteinfrom Zea mays W64 A, Nucleic Acids Res. 18(21):6426 (1990). See thefollowing site relating to the waxy promoter: Kloesgen, R. B., Gierl,A., Schwarz-Sommer, Z. S. and Saedler, H., Molecular analysis of thewaxy locus of Zea mays, Mol. Gen. Genet 203:237-244 (1986). Promotersthat express in the embryo, pericarp, and endosperm are disclosed inU.S. application Ser. Nos. 60/097,233 filed Aug. 20, 1998 and 60/098,230filed Aug. 28, 1998. The disclosures each of these are incorporatedherein by reference in their entirety.

[0064] Either heterologous or non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue.

[0065] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0066] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

[0067] The vector comprising the polynucleotide sequences useful in thepresent invention will typically comprise a marker gene that confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic or herbicide resistance. Suitable genes includethose coding for resistance to the antibiotic spectinomycin orstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance.

[0068] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to s inhibitaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron.

[0069] Typical vectors useful for expression of nucleic acids in higherplants are well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol., 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl.Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.). A variety of plant viruses that can be employed asvectors are known in the art and include cauliflower mosaic virus(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0070] The viral replicase polynucleotide can be expressed in eithersense or anti-sense orientation as desired. In plant cells, it has beenshown that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat'l. Acad. Sci. (USA) 85:8805-809 (1988); andHiaft et al., U.S. Pat. No. 4,801,340.

[0071] Another method of suppression is sense suppression. For anexample of the use of this method to modulate expression of endogenousgenes see, Napoli et al., The Plant Cell 2:279-289 (1990) and U.S. Pat.No. 5,034,323. Another method of down-regulation of the protein involvesusing PEST sequences that provide a target for degradation of theprotein.

[0072] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

[0073] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences, report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B.,et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinkingto a target nucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203.Use of crosslinking in triple-helix forming probes was also disclosed byHome et al., J Am Chem Soc (1990) 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-strandedoligonucleotides has also been described by Webb and Matteucci, J AmChem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674;Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds tobind, detect, label, and/or cleave nucleic acids are known in the art.See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;5,256,648; and, 5,681,941.

[0074] Proteins useful in the present invention include proteins derivedfrom the native protein by deletion (so-called truncation), addition orsubstitution of one or more amino acids at one or more sites in thenative protein. In constructing variants of the proteins of interest,modifications will be made such that variants continue to possess thedesired activity.

[0075] For example, amino acid sequence variants of the polypeptide canbe prepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, New York); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

[0076] The present invention includes catalytically active polypeptides(i.e., enzymes). Catalytically active polypeptides will generally have aspecific activity of at least 20%, 30%, or 40%, and preferably at least50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% that ofthe native (non-synthetic), endogenous polypeptide. Further, thesubstrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40%, or 50%, that of thenative (non-synthetic), endogenous polypeptide; and more preferably atleast 60%, 70%, 80%, or 90%. Methods of assaying and quantifyingmeasures of enzymatic activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art.

[0077] The methods of the present invention can be used with any cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The transformed cells produce viral replicase protein.

[0078] Typically, an intermediate host cell will be used in the practiceof this invention to increase the copy number of the cloning vector.With an increased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired plant cells. Host cells that can be used in the practice ofthis invention include prokaryotes, including bacterial hosts such asEschericia coli, Salmonella typhimurium, and Serratia marcescens.Eukaryotic hosts such as yeast or filamentous fungi may also be used inthis invention. It preferred to use plant promoters that do not causeexpression of the polypeptide in bacteria.

[0079] Commonly used prokaryotic control sequences include promoterssuch as the beta lactamase (penicillinase) and lactose (lac) promotersystems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp)promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) andthe lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0080] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

[0081] In some aspects of the invention, viral replicase proteins areintroduced into a cell to increase cell division. Synthesis ofheterologous proteins in yeast is well known. See Sherman, F., et al.,Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982). Twowidely utilized yeast for production of eukaryotic proteins areSaccharomyces cerevisiae and Pichia pastors. Vectors, strains, andprotocols for expression in Saccharomyces and Pichia are known in theart and available from commercial suppliers (e.g., Invitrogen). Suitablevectors usually have expression control sequences, such as promoters,including 3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

[0082] The protein can be isolated from yeast by lysing the cells andapplying standard protein isolation techniques to the lysates. Themonitoring of the purification process can be accomplished by usingWestern blot techniques or radioimmunoassay of other standardimmunoassay techniques.

[0083] The proteins useful in the present invention can also beconstructed using non-cellular synthetic methods. Techniques for solidphase synthesis are described by Barany and Merrifield, Solid-PhasePeptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.;Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156 (1963), and Stewartet al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.,Rockford, Ill. (1984). Proteins of greater length may be synthesized bycondensation of the amino and carboxy termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxy terminal end(e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

[0084] The proteins useful in this invention may be purified tosubstantial purity by standard techniques well known in the art,including detergent solubilization, selective precipitation with suchsubstances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. Detection of the expressed proteinis achieved by methods known in the art, for example, radioimmunoassays,Western blotting techniques or immunoprecipitation.

[0085] Expressing viral replicase polypeptides is expected to provide apositive growth advantage and increase crop yield.

[0086] In a preferred embodiment, the invention can be practiced in awide range of plants such as monocots and dicots. In a especiallypreferred embodiment, the methods of the present invention are employedin corn, soybean, sunflower, safflower, potato, tomato, sorghum, canola,wheat, alfalfa, cotton, rice, barley and millet.

[0087] The method of transformation/transfection is not critical to theinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform hostcells they may be directly applied. Accordingly, a wide variety ofmethods have been developed to insert a DNA sequence into the genome ofa host cell to obtain the transcription and/or translation of thesequence to effect phenotypic changes in the organism. Thus, any methodthat provides for efficient transformation/transfection may be employed.

[0088] A DNA sequence coding for the desired polynucleotide useful inthe present invention, for example a cDNA, RNA or a genomic sequence,will be used to construct an expression cassette that can be introducedinto the desired plant. Isolated nucleic acid acids of the presentinvention can be introduced into plants according techniques known inthe art. Generally, expression cassettes as described above and suitablefor transformation of plant cells are prepared.

[0089] Methods for transforming various host cells are disclosed inKlein et al. “Transformation of microbes, plants and animals by particlebombardment”, Bio/Technol. New York, N.Y., Nature Publishing Company,March 1992, v. 10 (3) pp. 286-291.

[0090] Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical, scientific, andpatent literature. See, for example, Weising et al., Ann. Rev. Genet.22: 421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG-mediated transfection, particle bombardment,silicon fiber delivery, or microinjection of plant cell protoplasts orembryogenic callus. See, e.g., Tomes et al., Direct DNA Transfer intoIntact Plant Cells Via Microprojectile Bombardment. pp.197-213 in PlantCell, Tissue and Organ Culture, Fundamental Methods. eds. O. L. Gamborgand G. C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995.Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0091] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327:70-73 (1987).

[0092]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maizeis described in U.S. Pat. No. 5,550,318.

[0093] Other methods of transformation include (1) Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, Vol. 6, P W J Rigby, Ed., London, AcademicPress, 1987; and Lichtenstein, C. P., and Draper, J,. In: DNA Cloning,Vol. 11, D. M. Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use ofA. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353, 1984), (3) the vortexingmethod (see, e.g., Kindle, Proc. Natl. Acad. Sci., USA 87:1228, (1990).

[0094] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingnucleic acids can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena et al., Nature, 325:274 (1987).DNA can also be injected directly into the cells of immature embryos andthe rehydration of desiccated embryos as described by Neuhaus et al.,Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in ProceedingsBio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).

[0095] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

[0096] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell, 2:603-618 (1990).

[0097] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

[0098] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.,80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0099] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). For maize cell culture andregeneration see generally, The Maize Handbook, Freeling and Walbot,Eds., Springer, New York (1994); Corn and Corn Improvement, ₃rd edition,Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis.(1988).

[0100] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed.

[0101] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

[0102] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated viral replicase nucleic acid. Progeny and variants, and mutantsof the regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introduced nucleicacid sequences.

[0103] Transgenic plants expressing a selectable marker can be screenedfor transmission of the viral replicase nucleic acid, for example,standard immunoblot and DNA detection techniques. Transgenic lines arealso typically evaluated on levels of expression of the heterologousnucleic acid. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can then analyzedfor protein expression by Western immunoblot analysis using thespecifically reactive antibodies of the present invention. In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0104] Plants that can be used in the method of the invention varybroadly and include monocotyledonous and dicotyledonous plants.Preferred plants include corn, soybean, sunflower, sorghum, canola,wheat, alfalfa, cofton, rice, barley, potato, tomato, and millet.

[0105] Seeds derived from plants regenerated from transformed plantcells, plant parts or plant tissues, or progeny derived from theregenerated transformed plants, may be used directly as feed or food, orfurther processing may occur.

[0106] Expression of the viral replicase nucleic acids in plants, suchas maize, is expected to enhance growth and biomass accumulation. Othermore specialized applications exist for these nucleic acids at the wholeplant level.

[0107] The present invention will be further described by reference tothe following detailed examples.

[0108] It is understood, however, that there are many extensions,variations, and modifications on the basic theme of the presentinvention beyond that shown in the examples and description, which arewithin the spirit and scope of the present invention. All publications,patents, and patent applications cited herein are hereby incorporated byreference.

EXAMPLES Example 1 Replicase Constructs

[0109] The replicase gene was obtained from Joachim Messing in thevector pWI-1 1, and was re-designated P100. Using P100 as the source,the replicase structural gene was cloned into an intermediate vectorcontaining the 35S promoter and a 35S 3′ sequence (for expressionstudies in dicotyledonous species, such as tobacco; designated P101 madein the Larkins Lab, Univ. of Arizona). From this intermediate plasmid,the RepA structural gene and the 35S 3′ sequence were excised using therestriction enzyme NcoI and PstI, and cloned into P101 (gamma zeinpromoter::uidA::Gamma zein 3′ region; after the removal of the GUSstructural gene from P101 using NcoI/PstI). This resulted in a finalconstruct containing an expression cassette with a maize gamma zeinpromoter sequence (GZ), the RepA coding sequence, a 35S terminator and agamma zein 3′ sequence (GZ′). Thus, the expression cassette had theconfiguration GZ:: RepA::35S::GZ′P102.

[0110] A derivative of the pWI-11 vector, with both iudA (encoding GUSexpression) and rep gene expression being driven by the bi-directionalpromoter elements in the WDV long intergenic region (WDV-LIR) was alsoprovided by the Messing lab (pWI-GUS).

Example 2 Replicase results in increased transient expression ofco-delivered transgenes

[0111] The plasmids listed in Table I below were used to evaluate theinfluence of Rep on transient expression of co-delivered transgenes. TheSuperMAS promoter is that described by Ni et al., 1996,Sequence-specific interactions of wound-inducible nuclear factors withmannopine synthase 2′ promoter wound responsive elements, Plant Mol.Biol. 30:77-96. The visible marker genes, GUS (b-glucoronidase;Jefferson R. A., Plant Mol. Biol. Rep. 5:387, 1987) and GFP (greenfluorescent protein; Chalfie et al., Science 263:802, 1994) have beendescribed, as has the maize-optimized GFP (GFPm; see copending U.S.patent application WO 97/41228). The Ubiquitin promoter has beendescribed (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992), as have thepinII (An et al., 1989, Plant Cell 1:115-122) and 35S (Odell et al.,1985, Nature 313:810-812) 3′ regions used in these expression cassettes.TABLE I Constructs used to evaluate the effect of replicase expressionon transient expression of co-delivered transgenes. Plasmid DescriptionP103 SuperMAS::GUS::pinll 3′ region P104 UBI::moPAT::CaMV35S 3′ regionP105 UBI::GFPm::pinll P100 WDV-LIR promoter::replicase

[0112] GFP expression in Maize

[0113] Transformation of the Rep plasmid DNA, P100, into the PioneerHi-Bred Int'l. Inc. proprietary inbred, N38, followed a well-establishedbombardment transformation protocol used for introducing DNA into thescutellum of immature maize embryos (Songstad, D. D. et al., In VitroCell Dev. Biol. Plant 32:179-183, 1996). It is noted that the anysuitable method of transformation can be used, such asAgrobacterium-mediated transformation and many other methods. Cells weretransformed by culturing maize immature embryos (approximately 1-1.5 mmin length) onto medium containing N6 salts, Erikkson's vitamins, 0,69g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4-5 days of incubationin the dark at 28° C., embryos were removed from the first medium andcultured onto similar medium containing 12% sucrose. Embryos wereallowed to acclimate to this medium for 3 h prior to transformation. Thescutellar surface of the immature embryos was targeted using particlebombardment with either a UBI::GFPm::pinII plasmid+aUBI::maize-optimized PAT::pinII plasmid (P105,control treatment) or witha combination of the UBI::GFPm::pinII plasmid P104+ the replicaseplasmidP100. Embryos were transformed using the PDS-1000 Helium Gun fromBio-Rad at one shot per sample using 650PSI rupture disks. DNA deliveredper shot averaged at 0.0667 ug. An equal number of embryos per ear werebombarded with either the control DNA mixture or the Rep/GFP DNAmixture. Following bombardment, all embryos were maintained on standardmaize culture medium (N6 salts, Erikkson's vitamins, 0.69 g/l proline, 2mg/l 2,4-D, 3% sucrose) for 2-3 days and then evaluated for transientGFP expression.

[0114] In both experiments, greater numbers of cells on the scutellarsurface transiently expressed GFP in the replicase-containing treatment.In experiment #1 with genotype N38, a mean of only 12 cells per embryotransiently expressed GFP in the treatment without replicase, while inthe replicase-treated embryos the mean number of GFP-expressing cellswas almost 20-fold greater (see Table II below). In the secondexperiment (Table III below), transient GFP expression in thereplicase-containing treatments was approximately 6.5-fold greater thanin the control treatments (no replicase). TABLE II Maize Experiment #1:Transient GFP expression is stimulated by Replicase Genotype & TreatmentGFP-expressing Explant (plasmids used) cells/embryo* Mean N38 immatureP104, P100 165, 290, 233 embryos 413, 149, 148 N38 immature P104, P1051, 22, 13  12 embryos

[0115] TABLE III Maize Experiment #2: Transient GFP expression Isstimulated by Replicase Genotype & Treatment GFP-expressing Explant(plasmids used) cells/embryo* Mean N38 immature P104, P100 1122, 108,285, 358 embryos (insert) 27, 249 N38 immature P104, P105 240, 10, 11,0, 0  52 embryos

[0116] Soybean

[0117] Tissue was excised from coyledons and placed on MS-based medium.A mixture of plasmid DNA, containing equal amounts of aSuperMas::GUS::pinII plasmid (P103) and the WDV-LIR::replicase plasmid(P100) was delivered into cells on the surface of the colyledon explantsusing particle-mediated delivery similar to that descibed for maizeabove. As a control, SuperMas::GUS::pinII plasmid(P103)+UBI::moPAT::CaMV35S (P105) was introduced into the same targetcells using an equal number of cotyledonary tissue pieces.

[0118] In the replicase-treatment, greater numbers of transientlyexpressing cells were observed on the cotyledon after GUS staining (seeFIG. 1). In addition, for cells exhibiting transient gene expression,the level of expression as judged by relative intensity of histochemicalstaining appeared greater in replicase-treated tissues (as compared tocontrols).

Example 3 RepA increases growth rates in early-developing stable maizetransformants

[0119] Transformation of the RepA plasmid DNA (P102), P102in Hi-IIfollowed the standard Hi-II bombardment transformation protocol(Songstad D. D. et al., In Vitro Cell Dev. Biol. Plant 32:179-183,1996). Cells were transformed by culturing maize immature embryos(approximately 1-1.5 mm in length) onto 560P medium containing N6 salts,Erikkson's vitamins, 0,69 g/l proline, 2 mg/l 2,4-D and 3% sucrose.After 4-5 days of incubation in the dark at 28° C., embryos were removedfrom 560P medium and cultured, scutellum up, onto 560Y medium which isequivalent to 560P but contains 12% sucrose. Embryos were allowed toacclimate to this medium for 3 h prior to transformation. The scutellarsurface of the immature embryos was targeted using particle bomardmentwith either a UBI::moPAT-GFPm::pinII plasmid (P106 alone as a controltreatment) or with a combination of the UBI::moPAT-GFPm::pinII plasmid(P106)+the GZ::RepA::35S:GZ′ plasmid (P102). Embryos were transformedusing the PDS-1000 Helium Gun from Bio-Rad at one shot per sample using650PSI rupture disks. DNA delivered per shot averaged at 0.0667 ug. Anequal number of embryos per ear were bombarded with either the controlDNA (PAT-GFP) or the RepA/PAT-GFP DNA mixture. Following bombardment,all embryos were maintained on 560L medium (N6 salts, Eriksson'svitamins, 0.5 mg/l thiamine, 20 g/l sucrose, 1 mg/l 2,4-D, 2.88 g/lproline, 2.0 g/l gelrite, and 8.5 mg/l silver nitrate). After 2-7 dayspost-bombardment, all the embryos from both treatments were transferredonto N6-based medium containing 3 mg/l bialaphos Pioneer 560P mediumdescribed above, with no proline and with 3 mg/l bialaphos). Plates weremaintained at 28° C. in the dark and were observed for colony recoverywith transfers to fresh medium occurring every two weeks. Two weeksafter DNA delivery, the newly-forming callus was examined usingepifluorescence under the dissecting microscope (usingcommercially-available filter combinations for GFP excitation andemission).

[0120] At 2 weeks post-bombardment, numerous cells on the surface of thescutellar-derived tissue were expressing GFP in the control treatment(no RepA), but all expressing foci consisted of single cell. Nomulticellular GFP-expressing clusters were observed in the control. Atthis same time-point, 2-weeks after DNA-delivery, the same sprinkling ofsingle-celled GFP-expressing foci were observed on the surface of thetissue that had received the RepA/PAT-GFP mixture. However, numerousmacroscopic GFP-expressing multicellular clusters were also apparent(see FIG. 2). Many embryos were observed with multiple transgenicmicrocalli developing on the surface, with as many as 7 apparently-independent transformants beginning to grow from a single embryo (thishas never been reported before for particle bombardment of maize).

[0121] After 3 weeks, GFP-expressing single cells could still beobserved in both treatments, although the frequency had declined. In thecontrol treatment, a solitary GFP-expressing multicellular colony weobserved to be developing on one embryo (out of 50 total). In theRepA-treated embryos, the growth rate of the developing transgenic callicontinued to be very rapid. Many of the multiple colonies apparentlygrowing from single embryos were already in danger of co-mingling bygrowing together into a single mass. Many colonies were picked off theembryos to grow them separately. At 5 weeks post-bombardment, many RepAcolonies continued to grow rapidly (some may have been too small tosurvive independently). While growing rapidly, these RepA-treatedtransgenic calli maintain a healthy embryogenic character.

Example 4 RepA increases cell division rates in tobacco suspensionculture cells

[0122] For tobacco BY-2 suspension culture cells, the followingconstruct was used; 35S promoter::RepA::35S 3′ region (P101). Suspensioncells were grown in a medium comprised of Murashige and Skoog salts(Life Technologies, Inc., Grand Island, N.Y.), 100 mg/l inositol, 1 mg/lthiamine, 180 mg/l KH2PO4, 30 g/l sucrose, and 2 mg/l 2,4-D, subculturedevery 7-10 days, and grown on a gyratory shaker at 150 RPM, 24° C. inthe dark. Three days after subculturing, cells were pipetted ontosolidified agar medium for bombardment, and left in the dark for 24hours. Bombardment was performed using a BioRad PDS-1000, using heliumat 650 PSI and 25 inches Hg, with 8 cm distance between the stoppingplate and petri dish. All cells were shot once with 500 ng gold and 0.5μg DNA. All the treated cells received a plasmid containing a35S::GFP::35S expression cassette (P108), with half receiving anadditional plasmid containing the 35S::RepA::35S cassette. Afterbombardment, the cells were monitored for GFP expression and celldivision.

[0123] After 24 hours, GFP-expressing cells were scored as non-dividing(single fluorescent cells) or as having divided during the intervening24-hour period (i.e. GFP-expressing doublets with the characteristicnewly-formed division plate between the two fluorescent daughter cells).For the control treatment (GFP alone), 37.5% (with a standard error of1.8, calculated for three replicates) of the total number ofGFP-expressing cells had undergone division during this period. In thetreatment where GFP+RepA expression cassettes were introducedsimultaneously, the percentage of GFP-expressing cells that hadundergone division increased substantially to 45.7 (SE=5.7).

Example 5 RepA increases maize transformation frequency

[0124] For transformation experiments, a construct was used in which theRepA coding sequence was cloned into a maize expression cassette (P102,described above). Delivery of the RepA gene in an appropriate plantexpression cassette (for example, in a GZ::RepA::35S:GZ-containingplasmid) along with marker gene cassettes was accomplished usingparticle bombardment. DNA was introduced into maize cells capable ofgrowth on suitable maize culture medium (freshly isolated immatureembryos). See Table IV below for treatments. Immature embryos of theHi-II genotype were used as the target for co-delivery of plasmids. Toassess the effect on transgene integration, growth ofbialaphos-resistant colonies on selective medium was a reliable assay.Within 1-7 days after DNA introduction, the embryos were moved ontoculture medium containing 3 mg/l of the selective agent bialaphos.Embryos, and later callus, were transferred to fresh selection platesevery 2 weeks. Four-six weeks after bombardment, bialaphos-resistantcalli were scored and transferred to separate plates to prevent mixingof transformants as they continue to grow. Expression of the visiblescorable marker ( GUS or GFP) was used to confirm transformation. In theRepA-treated embryos, higher numbers of stable transformants wererecovered (likely a direct result of increased integration frequencies).TABLE IV Experimental design for assessing the influence of RepAexpression on recovery of stable maize transformants. Experiment #Control Treatment 1 & 2 None included in E35S::bar::pinll +UBI::GUS::pinll + experiment GZ::RepA::35S:GZ′ 3 UBI::PATm-GFPm::pinllUBI::PATm-GFPm::pinll + GZ::RepA::35S:GZ′

[0125] Experiment #1. This experiment was originally designed to testRepA expression in endosperm. Thus, we used all of the embryos from theavailable Hi-II ears on this day to introduce RepA along with the markergenes (P107 the construct containing Enhanced-35S promoter::bar::pinIIand UBI::GUS::pinII). The frequencies for Hi-II transformation usingP107 alone (E35S::bar::pinII+UBI::GUS::pinII) during this period wereaveraging between 2-3%, providing a good basis of comparison. In thisexperiment, our transformation frequency with P107+GZ::RepA (P102) was8.8% (33 transformants /375 starting embryos).

[0126] Experiment #2. Again, the original intent of this experiment wasto generate endosperm-expressing RepA transformants (not to comparetransformation frequencies). As in the first experiment, the observedresult was unexpected; transformation frequency using P107+GZ::RepA(P102) was 29.2% (73 transformants /250 starting embryos). Thisrepresented approximately a 10-fold increase over the 2-3%transformation frequencies observed in other experiments conductedduring this period using similar marker genes (the bar gene to conferbialaphos resistance and GUS as a visible marker).

[0127] Experiment #3. In this experiment, numerous ears were used.Immature embryos were isolated from each ear, randomized on plates andthen split between each of the two treatments (+/−RepA). This comparisonused a total of 725 embryos per treatment, harvested from a total of 29ears (25 embryos/ear/treatment). Transformation frequencies werecalculated on a per-ear basis and then expressed as the mean. MeanTransformation Standard Treatment Frequency (%) DeviationUBI::PATm-GFPm::pinll (Control)  2.2 1.8 UBI::PATm-GFPm::pinll + 17.08.5 GZ::RepA::35S:GZ′

[0128] This tightly controlled experiment validated the preliminaryresults in Experiments #1 & #2. Across many replicates (individual earsharvested on separate dates), the mean frequency for RepA-treatedimmature embryos was over 7.5-fold greater than for embryos treatedsolely with the control plasmid. For particle-mediated transformation ofHi-II immature embryos, this is a remarkable improvement intransformation frequency. The calli recovered from the RepA treatmentsgrew vigorously, were embryogenic, and easily regenerated into plants.Plants regenerated to date have appeared phenotypically normal, wereboth male and female fertile, and transmitted the transgenes (and theirexpression) to progeny in expected Mendelian ratios.

Example 6 RepA alters the cell cycle phenotype in cell populations fromtransgenic calli

[0129] Transformation of Hi-II immature embryos was performed using theprottocol described in Example 3. A mixture of plasmid DNA, containingequal amounts of a E35S::bar::pinII+UBI::GUS::pinII plasmid (P107) and aGZ::RepA35S::GZ′ plasmid (P102), was delivered into scutellar cells ofthe immature embryos using particle-mediated delivery. As a control,E35S::bar::pinII+UBI::GUS::pinII (P107) plasmid alone was introducedinto the same target cells on the surface of the scutellum for an equalnumber of embryos. One week after particle bombardment, all the embryosfrom both treatments were transferred onto N6-based medium containing 3mg/l bialaphos. After 6 weeks, stable transformants were scored, andexpression of a second marker gene (GUS) was used to confirm thetransgenic nature of the callus. Transgenic callus expressing bar andGUS alone (from the control treatment), or transgenic callus expressingbar, GUS and RepA were used to isolate nuclei. For extraction of nuclei,callus was macerated with a straight-edge razor blade in a bufferconsisting of 45 mM CgCL₂, 30 mM sodium citrate, 20 mM MOPS buffer, 0.1%v/v Triton X 100. For each callus event sampled, tissue (approximately 1cm³) was transferred to a Petri dish, and macerated with a small volumeof the chopping buffer. The resulting suspension was then passedsequentially through 60 um and 20 um sieves and transferred to a 15 mlcentrifuge tube on ice. Tubes were centrifuged at 100 g for 5 minutes at4° C. The supernatant was decanted, the pellets resuspended in ˜750 μlof staining solution (100 μg/ml propidium iodide in chopping buffer) andtransferred to tubes for analysis in the flow cytometer. Stained nucleiwere analyzed on an EPICS-XL-MCL flow cytometer using a 488 nm argonlaser for excitation and measuring emission from 500-550 nm. Collectingpropidium iodide fluorescence measurements on a per-nucleus basis(equivalent to the DNA content per nucleus) permitted the assessment ofcell cycle stages in the callus-cell population.

[0130] The cell cycle profile from the control callus was typical ofmaize callus cell populations, with a predominant G1 peak (approximately80%), a low percentage of S phase (8%), and a low percentage of G2(approximately 12%). In a RepA-treated callus transformant, the cellcycle profile was dramatically shifted, with approximately 7% G1, 8% Sphase and 85% in the G2 phase (see FIG. 3).

Example 7 Transient RepA activity enhances transformation frequency

[0131] For this specific application (using transient RepA-mediated cellcycle stimulation to increase transient integration frequencies), it maybe desirable to reduce the likelihood of ectopic stable expression ofthe RepA gene. Strategies for transient-only expression can be used.This includes delivery of RNA (transcribed from the RepA gene),chemically end-modified DNA expression cassettes that typically will notintegrate, or RepA protein along with the transgene cassettes to beintegrated to enhance transgene integration by transient stimulation ofcell division. Using well-established methods to produce RepA-RNA, thiscan then be purified and introduced into maize cells using physicalmethods such as microinjection, bombardment, electroporation or silicafiber methods. For protein delivery, the gene is first expressed in abacterial or baculoviral system, the protein purified and thenintroduced into maize cells using physical methods such asmicroinjection, bombardment, electroporation or silica fiber methods.Alternatively, RepA proteins are delivered from Agrobacteriumtumefaciens into plant cells in the form of fusions to Agrobacteriumvirulence proteins. Fusions are constructed between RepA and bacterialvirulence proteins such as VirE2, VirD2, or VirF which are known to bedelivered directly into plant cells. Fusions are constructed to retainboth those properties of bacterial virulence proteins required tomediate delivery into plant cells and the RepA activity required forenhancing transgene integration. This method ensures a high frequency ofsimultaneous co-delivery of T-DNA and functional RepA protein into thesame host cell. The methods above represent various means of using theRepA gene or its encoded product to transiently stimulate DNAreplication and cell division, which in turn enhances transgeneintegration by providing an improved cellular/molecular environment forthis event to occur.

Example 8 Altering RepA expression stimulates the cell cycle and growth

[0132] Based on our observations, expression of RepA genes increasescell division rates. Increases in division rate are assessed in a numberof different manners, being reflected in smaller cell size, more rapidincorporation of radiolabeled nucleotides, and faster growth (i.e. morebiomass accumulation). Delivery of the RepA in an appropriate plantexpression cassette is accomplished through numerous well- establishedmethods for plant cells, including for example particle bombardment,sonication, PEG treatment or electroporation of protoplasts,electroporation of intact tissue, silica-fiber methods, microinjectionor Agrobacterium-mediated transformation. The result of RepA geneexpression will be to stimulate the G1/S transition and hence celldivision, providing the optimal cellular environment for integration ofintroduced genes (as per Example 1). This will trigger a tissue cultureresponse (cell divisions) in genotypes that typically do not respond toconventional culture techniques, or stimulate growth of transgenictissue beyond the normal rates observed in wild-type (non-transgenic)tissues. To demonstrate this, the RepA gene is cloned into a cassettewith a constitutive promoter (i.e. either a strong maize promoter suchas the ubiquitin promoter including the first ubiquitin intron, or aweak constitutive promoter such as nos). Either particle-mediated DNAdelivery or Agrobacterium-mediated delivery are used to introduce theGZ::RepA::35S:GZ-containing plasmid along with aUBI::bar:pinII-containing plasmid into maize cells capable of growth onsuitable maize culture medium. Such competent cells can be from maizesuspension culture, callus culture on solid medium, freshly isolatedimmature embryos or meristem cells. Immature embryos of the Hi-IIgenotype are used as the target for co-delivery of these two plasmids,and within 1-7 days the embryos are moved onto culture medium containing3 mg/l of the selective agent bialaphos. Embryos, and later callus, aretransferred to fresh selection plates every 2 weeks. After 6-8 weeks,transformed calli are recovered. In treatments where both the bar geneand RepA gene have been transformed into immature embryos, a highernumber of growing calli are recovered on the selective medium and callusgrowth is stimulated (relative to treatments with the bar gene alone).When the RepA gene is introduced without any additional selectivemarker, transgenic calli can be identified by their ability to grow morerapidly than surrounding wild-type (non-transformed) tissues. Transgeniccallus can be verified using PCR and Southern analysis. Northernanalysis can also be used to verify which calli are expressing the bargene, and which are expressing the maize RepA gene at levels abovenormal wild-type cells (based on hybridization of probes to freshlyisolated mRNA population from the cells).

[0133] Inducible Expression

[0134] The RepA gene can also be cloned into a cassette with aninducible promoter such as the benzenesulfonamide-inducible promoter.The expression vector is co-introduced into plant cells and afterselection on bialaphos, the transformed cells are exposed to the safener(inducer). This chemical induction of RepA expression results instimulated G1/S transition and more rapid cell division. The cells arescreened for the presence of RepA RNA by northern, or RT-PCR (usingtransgene specific probes/oligo pairs), for RepA-encoded protein usingRepA-specific antibodies in Westerns or using hybridization. IncreasedDNA replication is detected using BrdU labeling followed by antibodydetection of cells that incorporated this thymidine analogue. Likewise,other cell cycle division assays could be employed, as described above.

Example 9 Control of RepA gene expression using tissue-specific orcell-specific promoters provides a differential growth advantage

[0135] RepA gene expression using tissue-specific or cell-specificpromoters stimulates cell cycle progression in the expressing tissues orcells. For example, using a seed-specific promoter will stimulate celldivision rate and result in increased seed biomass. Alternatively,driving RepA expression with an tassel-specific promoter will enhancedevelopment of this entire reproductive structure.

[0136] Expression of RepA genes in other cell types and/or at differentstages of development will similarly stimulate cell division rates.Similar to results observed in Arabidopsis (Doerner et al., 1996),root-specific or root-preferred expression of RepA will result in largerroots and faster growth (i.e. more biomass accumulation).

Example 10 Meristem Transformation

[0137] Meristem transformation protocols rely on the transformation ofapical initials or cells that can become apicial initials followingreorganization due to injury or selective pressure. The progenitors ofthese apical initials differentiate to form the tissues and organs ofthe mature plant (i.e. leaves, stems, ears, tassels, etc.). Themeristems of most angiosperms are layered with each layer having its ownset of initials. Normally in the shoot apex these layers rarely mix. Inmaize the outer layer of the apical meristem, the L1, differentiates toform the epidermis while descendents of cells in the inner layer, theL2, give rise to internal plant parts including the gametes. Theinitials in each of these layers are defined solely by position and canbe replaced by adjacent cells if they are killed or compromised.Meristem transformation frequently targets a subset of the population ofapical initials and the resulting plants are chimeric. If for example, 1of 4 initials in the L1 layer of the meristem are transformed only ¼ ofepidermis would be transformed. Selective pressure can be used toenlarge sectors but this selection must be non-lethal since large groupsof cells are required for meristem function and survival. Transformationof an apical initial with a RepA expression cassette under theexpression of a promoter active in the apical meristem (either meristemspecific or constitutive) would allow the transformed cells to growfaster and displace wildtype initials driving the meristem towardshomogeneity and minimizing the chimeric nature of the plant body. Todemonstrate this, the RepA gene is cloned into a cassette with apromoter that is active within the meristem (i.e. a promoter active inmeristematic cells such as the maize histone, cdc2 or actin promoter).Coleoptilar stage embryos are isolated and plated meristem up on a highsucrose maturation medium (see Lowe et al., 1997). The RepA expressioncassette along with a reporter construct such as Ubi:GUS:pinII can thenbe co-delivered (preferably 24 hours after isolation) into the exposedapical dome using conventional particle gun transformation protocols. Asa control the RepA construct can be replaced with an equivalent amountof pUC plasmid DNA. After a week to 10 days of culture on maturationmedium the embryos can be transferred to a low sucrose hormone-freegermination medium. Leaves from developing plants can be sacrificed forGUS staining. Transient expression of the RepA gene in meristem cells,through stimulation of the G1→S transition, will result in greaterintegration frequencies and hence more numerous transgenic sectors.Integration and expression of the RepA gene will impart a competitiveadvantage to expressing cells resulting in a progressive enlargement ofthe transgenic sector. Due to the enhanced growth rate inRepA-expressing meristem cells, they will supplant wild-type meristemcells as the plant continues to grow. The result will be bothenlargement of transgenic sectors within a given cell layer (i.e.periclinal expansion) and into adjacent cell layers (i.e. anticlinalinvasions). As an increasingly large proportion of the meristem isoccupied by RepA-expressing cells, the frequency of RepA germlneinheritance goes up accordingly.

Example 11 Use of Flp/Frt system to excise the RepA cassette

[0138] In cases where the RepA gene has been integrated and RepAexpression is useful in the recovery of maize trangenics, but isultimately not desired in the final product, the RepA expressioncassette (or any portion thereof that is flanked by appropriate FRTrecombination sequences) can be excised using FLP-mediated recombination(see co-pending U.S. Patent Application US 98/24640).

What is claimed is:
 1. A method for increasing transformationfrequencies in a target plant cell comprising introducing into thetarget plant cell an isolated plant geminivirus replicase polypeptide.2. The method of claim 2 wherein the geminivirus replicase polypeptideis wheat dwarf virus Replicase.
 3. The method of claim 3 wherein thegeminivirus replicase polypeptide is RepA.
 4. The method of claim 1further comprising introducing into the target plant cell apolynucleotide of interest.
 5. The method of claim 4 wherein the plantcell is from a monocot or a dicot plant.
 6. The method of claim 5wherein the plant cell is from corn, soybean, sunflower, sorghum,canola, wheat, alfalfa, cotton, rice, barley, potato, tomato, andmillet.
 7. A method for providing a positive growth advantage in atarget plant cell comprising introducing into the target plant cell anisolated plant geminivirus replicase polypeptide.
 8. The method of claim7 wherein the geminivirus replicase polypeptide is wheat dwarf virusReplicase.
 9. The method of claim 8 wherein the geminivirus replicasepolypeptide is RepA.
 10. The method of claim 7 wherein the plant cell isa monocot or a dicot.
 11. The method of claim 10 wherein the plant cellis from corn, soybean, sunflower, sorghum, canola, wheat, alfalfa,cotton, rice, barley, potato, tomato, and millet.
 12. A method formodulating cell division of a target plant cell capable of dividing,comprising introducing into the target plant cell an isolated viralreplicase polypeptide.
 13. The method of claim 12 wherein thepolypeptide is wheat dwarf virus Replicase.
 14. The method of claim 13wherein the polypeptide is RepA.
 15. The method of claim 12 wherein theplant cell is from a monocot or a dicot plant.
 16. The method of claim15 wherein the plant cell is from corn, soybean, sunflower, sorghum,canola, wheat, alfalfa, cotton, rice, barley, potato, tomato, andmillet.